PPR ssDNA-Binding Domains for Programmable Gene Regulation

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Solution Overview

Problem

Existing technologies lack the ability to manipulate single-stranded DNA (ssDNA) effectively due to the insufficiency of natural ssDNA-binding proteins, which complicates the precise targeting and manipulation of this vulnerable nucleic acid form, crucial for processes like gene regulation and telomere maintenance.

Innovation Solution

Development of consensus artificial Pentatricopeptide Repeat (cPPR) domains that can be designed to bind ssDNA in a specific, modular, and programmable manner, allowing for targeted regulation of gene expression by inhibiting human telomerase activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If natural ssDNA-binding proteins are used, then ssDNA binding capability is achieved, but sequence specificity and programmability are insufficient

Engineering Contradiction:

Improvesequence specificityVSAvoidprogrammability

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The protein is divided into multiple independent PPR repeat units (typically 2-30 repeats), each capable of binding specific nucleotide sequences. This segmentation allows modular design where individual repeats can be independently engineered to recognize different sequences, achieving both high specificity and programmability through combinatorial arrangement of functional units.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The invention changes the binding specificity parameters by modifying amino acid residues at specific positions (positions 5 and 35) within each PPR repeat. By altering these key positions, the protein can be programmed to bind different nucleotide sequences, transforming a naturally limited binder into a programmable tool with customizable specificity.

Inventive Principle:

Principle #35Parameter changes

2Adaptability or versatility

If PPR proteins are designed for RNA binding, then RNA recognition capability is achieved, but ssDNA binding capability is insufficient

Engineering Contradiction:

Improvenucleic acid binding capabilityVSAvoidssDNA binding reliability

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The PPR protein scaffold is designed to perform multiple functions: it can bind both RNA and DNA nucleic acids, and can be programmed to recognize different sequences within each class. This universality is achieved through the conserved structural framework of PPR repeats that maintains the ability to interact with nucleic acid backbones while allowing sequence-specific recognition through programmable amino acid changes.

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Ease of operation

If existing ssDNA manipulation technologies are used, then some ssDNA binding is achieved, but precise targeting and manipulation capability is insufficient

Engineering Contradiction:

Improvemanipulation capabilityVSAvoidtargeting precision

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The PPR protein system provides dynamic programmability where the binding specificity can be adjusted by changing the amino acid sequence of individual repeats. This dynamic reconfigurability allows the same protein scaffold to be adapted for different target sequences and applications, enabling precise targeting of specific ssDNA locations while maintaining ease of manipulation through modular design.

Inventive Principle:

Principle #15Dynamics

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

The cPPR domains enable precise binding to any telomere sequence, providing a mechanism for regulating gene expression and blocking telomere extension by human telomerase, offering potential therapeutic applications in cancer treatment.

Implementation Method 1

PPR proteins that can bind ssDNA in a sequence-specific manner analogous to how they bind RNA

Methodology Applied

Scientific EffectMolecular recognition:

Data Source

PatentUS12441770B2Proteins and their use for nucleotide binding

Publication Date: 2025.10.14 THE UNIVERSITY OF WESTERN AUSTRALIA

AI summary

A method of regulating expression of a gene in a cell is described, comprising the step of introducing into the cell a recombinant polypeptide comprising a PPR ssDNA-binding domain which itself comprises at least a pair of PPR ssDNA base-binding motifs. The PPR ssDNA base-binding motifs of the PPR ssDNA-binding domain are operably capable of binding the target ssDNA. Recombinant polypeptides comprising at least one PPR sDNA-binding domain capable of binding to target ssDNA sequence are also described, together with fusion proteins comprising the recombinant PPR ssDNA-binding domains, as well as isolated nucleic acids useful in preparing the recombinant polypeptides described. Recombinant vectors; compositions comprising the recombinant polypeptides; isolated nucleic acids; recombinant vectors; host cells comprising same; use of same in the manufacture of a medicament for regulating gene expression; as well as systems and kits for regulating gene expression are also described.